To design therapy against cancer, it is desirable to seek molecular targets of cancer or cancer stem cells that are absent from normal cells. Aberrant glycosylation is often associated with tumor progression and was first described by Meezan et al. in 1969 with the demonstration that cancer glycans differ from healthy cells. (Meezan E, et al. (1969) Biochemistry 8:2518-2524.) Aberrant glycosylations include loss or over-expression of certain structures, the persistence of truncated structures and the emergence of novel structures. The structural differences were later supported by many histological evidences using lectin-staining compared with healthy and malignant tissue. (Turner G A (1992) Clin Chim Acta 208:149-171; Gabius H J (2000) Naturwissenschaften 87:108-121.)
More recently, tumor associated carbohydrate antigens were identified by monoclonal antibodies and mass spectrometry. (Shriver Z, et al. (2004) Nat Rev Drug Disc 3:863-873; Pacino G, et al. (1991) Br J Cancer 63:390-398.) To date, numerous tumor associated antigens expressed on cancer cells in the form of glycolipids or glycoproteins have been characterized and correlated to certain types of cancers. (Bertozzi C R, Dube D H (2005) Nat Rev Drug Discovery 4:477-488.) Although relatively little is known about the role of surface carbohydrates play in malignant cells, passively administered or vaccine induced antibodies against these antigens have correlated with improved prognosis.
Of the tumor associated glycans reported, the glycolipid antigen Globo H (Fucα1→2 Galβ1→3 GalNAcβ1→3 Galα1→4 Galβ1→4 Glc) was first isolated and identified in 1984 by Hakomori et al. from breast cancer MCF-7 cells. (Bremer E G, et al. (1984) J Biol Chem 259:14773-14777.) Further studies with anti-Globo H monoclonal antibodies showed that Globo H was present on many other cancers, including prostate, gastric, pancreatic, lung, ovarian and colon cancers and only minimal expression on luminal surface of normal secretory tissue which is not readily accessible to immune system. (Ragupathi G, et al. (1997) Angew Chem Int Ed 36:125-128.) In addition, it has been established that the serum of breast cancer patient contains high level of anti-Globo H antibody. (Gilewski T et al. (2001) Proc Natl Acad Sci USA 98:3270-3275; Huang C-Y, et al. (2006) Proc Natl Acad Sci USA 103:15-20; Wang C-C, et al. (2008) Proc Natl Acad Sci USA 105(33):11661-11666) and patients with Globo H-positive tumors showed a shorter survival in comparison to patients with Globo H-negative tumors. (Chang, Y-J, et al. (2007) Proc Natl Acad Sci USA 104(25):10299-10304.) These findings render Globo H, a hexasaccharide epitope, an attractive tumor marker and a feasible target for cancer vaccine development.
Globo H is a cancer antigen overly expressed in various epithelial cancers. It has been suggested that this antigen can serve as a target in cancer immunotherapy. While vaccines have been developed to elicit antibody responses against Globo H, their anti-cancer efficacies are unsatisfactory due to low antigenicity of Globo H. There is a need for a new vaccine capable of eliciting high levels of immune responses targeting Globo H.
Stem cells are defined as a group of cells with the capacity for self-renewal and for differentiation into different types of cells and tissues. (Reya T et al., (2001) Nature 414:105-111.) As both malignant tumors and normal tissues contain heterogeneous populations of cells, cancer stem cells might play a key role in tumor growth and maintaining tumor heterogeneity. Cancer stem cells have been identified from a variety of solid tumors, such as brain, breast, colon, and prostate cancers. Breast cancer stem cells (BCSCs) were first shown to reside in the CD24−CD44+ subpopulation of breast cancer by Al-Hajj et al., based on their ability to generate tumors with phenotypic diversity on xenotransplantation into NOD/SCID mice (Al-Hajj M, et al., (2003) Proc Natl Acad Sci USA 100:3983-3988). The majority of early disseminated cancer cells in the bone marrow of breast cancer patients displayed the phenotype of CD24−CD44+ (Balic M et al., (2006) Clin Cancer Res 12:5615-5621), suggesting that BCSCs were capable of metastasis. Based on their capability for growth, differentiation, and metastasis and their resistance to radiation, BCSCs are a major target for therapy of breast cancer (Tang C. et al., (2007) FASEB J. 21:1-9).
In breast cancer, Globo H expression was observed in >60% of ductal, lobular, and tubular carcinoma, but not in nonepithelial breast tumors (Mariani-Constantini R et al., (1984) Am. J. Pathol. 115:47-56). Globo H is not expressed in normal tissue except for weak expression in the apical epithelial cells at lumen borders, a site that appears to be inaccessible to the immune system (Id.; Zhang S. et al., (1997) Int. J. Cancer 73:42-49).
Globo H also is expressed in breast cancer stem cells (BCSCs). Flow cytometry revealed Globo H is expressed in 25/41 breast cancer specimens (61.0%). Non-BCSCs from 25/25 and BCSCs from 8/40 (20%) express Globo H. The stage-specific embryonic antigen 3 (SSEA-3), the pentasaccharide precursor of Globo H, is expressed in 31/40 (77.5%) tumors. Non-BCSCs from 29/31 and BCSCs from 25/40 (62.5%) expressed SSEA-3. (Chang W-W. et al., (2008) Proc Natl Acad Sci USA 105(33):11667-11672.)
Danishefsky and Livingston previously reported the preparation of Globo H-KLH vaccine (Gilewski T et al. (2001) Proc Natl Acad Sci USA 98:3270-3275; Ragupathi G, et al. (1997) Angew Chem Int Ed 36:125-128; Kudryashov V, et al. (1998) Glycoconj J. 15:243-249; Slovin S F et al (1997) Proc Natl Acad Sci USA 96:5710-5715) and the heptavalent vaccine (containing GM2, Globo H, Lewis Y, Tn, STn, TF, and Tn-MUC1 individually conjugated to KLH; Sabbatini P J et al (2007) Clin Cancer Res 13:4170-4177) against a variety of cancers. However, patients immunized with the heptavalent vaccine induced antibody responses against only five of the seven antigens except GM2 and Lewis Y antibodies. Rather than ubiquitously expressed antigen such as GM2, Globo H exceptionally expressed on tumor cells with only minimal level on normal secretory tissue makes it a desirable target for vaccine development. In their studies, ozonolysis of Globo H aglycone was followed by reductive amination with KLH carrier protein to generate about 150 carbohydrate units per protein. (Ragupathi G, et al. (1997) Angew Chem Int Ed 36:125-128.) Further refinement increased the carbohydrate conjugation ratio to about 720:1 by using MMCCH linker. (Wang S-K, et al. (2008). Proc Natl Acad Sci USA 105:3690-3695.) However, it was difficult to precisely characterize the glycoconjugate. In addition, the synthetic vaccine in combination with the immunological adjuvant QS-21 was shown to induce mainly IgM and to a lesser extent IgG antibodies in both prostate and metastatic breast cancer patients. In the phase I clinical trial, the vaccine also showed minimal toxicity with transient local skin reactions at the vaccination site. (Gilewski T et al. (2001) Proc Natl Acad Sci USA 98:3270-3275; Ragupathi G, et al. (1997) Angew Chem Int Ed 36:125-128; Slovin S F et al (1997) Proc Natl Acad Sci USA 96:5710-5715.) Mild flu-like symptoms which have been observed in some of the patients were probably associated with the side effect of QS-21. A pentavalent vaccine containing five prostate and breast cancer associated carbohydrate antigens—Globo-H, GM2, STn, TF and Tn—conjugated to maleimide-modified carrier protein KLH has been reported to produce anti-Globo H sera with higher titers of IgG than IgM in ELISA assays. (Zhu J. et al. (2009) J. Am. Chem. Soc. 131(26):9298-9303).
Therefore, it is desirable to identify an alternative carrier and adjuvant to augment the antibody response to Globo H, especially with high titer of IgG, and to improve the vaccine efficacy with minimal side effects.